Application of molecular absorption properties of horseradish peroxidase for self-indicating enzymatic interactions and analytical methods

In this paper an in depth study is presented of the use of the horseradish peroxidase (HRP) enzyme as a self-indicating biorecognition reagent in UV-vis molecular absorption spectrometry. The HRP/H2O2 reaction mechanism in the absence of an external substrate has been clarified, and the interaction between HRP and glucose oxidase (GOx) has been studied. It has been demonstrated that GOx can act as a substrate of HRP; in both cases the kinetic constants have been obtained and mathematical models have been developed. Second, the HRP/H2O2 reaction is used to follow a H2O2-producing enzymatic reaction, the glucose reaction with GOx being used as a model. As an application of this, two methodologies have been proposed for glucose determination: with or without previous incubation of glucose with GOx. In both cases mathematical models relating HRP absorbance changes to glucose concentration have been developed and tested; both methods have been optimized, analytically characterized, and tested for glucose determination in samples. The methodology described could be applied to other heme-proteins and to other H2O2-producing enzymatic reactions. The models permit the reaction constants to be calculated. From the analytical chemistry point of view the models allow the prediction of the method sensitivity for other analytes involved in this type of reaction if the kinetic constants are known and can be used in the design of optical sensors.     

Application of time-of-flight mass spectrometry to the analysis of phototransformation products of diclofenac in water under natural sunlight

Exact mass capabilities of time-of-flight (TOF) mass spectrometry along with other mass spectrometric techniques have been evaluated to elucidate a complete range of dichlofenac phototransformation products. Photolysis experiments with diclofenac in water under direct solar irradiation were performed to characterise the main phototransformation products generated and to determine their stability. Photolysis experiments were performed in both demineralised water and reconstructed standard freshwater. Samples were extracted before analysis by solid phase extraction (SPE) with Oasis HLB and MAX cartridges. Separation and identification of the transformation products were accomplished by the combined use of gas chromatography-mass spectrometry (GC/MS) and liquid chromatography coupled with time-of-flight mass spectrometry (LC/TOFMS). Both techniques provided complementary information that enabled the identification of 13 phototransformation products. Six of them were identified by GC/MS through the structural information provided by the full scan mass spectra obtained under electron impact (EI) ionisation and the confirmation of the molecular mass provided by positive chemical ionisation (PCI) analyses. Accurate mass measurements obtained by LC/TOFMS provided the elucidation of seven polar transformation products. The low mass error observed (<2 ppm) enabled the assignment of highly probable empirical formulas as well as identification of a process dimerisation route. The photoproducts identified demonstrated that photolysis of diclofenac occurs by two main routes. One is the consequence of the initial photocyclisation of diclofenac into carbazole derivatives. The other route goes through the initial decarboxilation of diclofenac and further oxidation of the alkyl-chain, which are typical photolytic process reactions. The main photoproduct identified was 8-chloro-9H-carbazole-1yl-acetic acid.     

Indene and pseudoazulene discotic liquid crystals: a synthetic and structural study

Several new liquid-crystalline indene and pseudoazulene systems are reported. These molecules give rise to either columnar hexagonal mesophases and/or columnar plastic phases. The unique nature of these compounds stems from their non-classical discotic structure. Although the molecules have rigid aromatic cores, they lack terminal tails and instead the polarizable atoms (S, halogens) or polar groups (CN, CO) act as unusual soft parts. On the basis of many structurally related materials, we conclude that for this type of compound molecular stacking in the solid state is a prerequisite for the appearance of a columnar mesophase, although other intermolecular interactions within the layers are also important in establishing liquid-crystalline order. The behavior reported for these mesomorphic molecules opens up new possibilities in the search for related molecular interactions that might be useful for the construction of supramolecular architectures with particular properties.     

Capillary attraction of colloidal particles at an aqueous interface

We study the capillary forces arising from charged colloidal particles trapped at an oil-water interface. Since it is quadratic in the electric field, the electric stress acting on the interface cannot be written as the superposition of one-particle terms. Indeed, we find that the interfacial pressure is dominated by two-particle terms, which induce capillary forces involving one, two, three, or four particles. The dominant interaction is attractive and varies with the inverse cube of the particle distance.     

Synthesis and molecular structure of methyl 4-O-methyl-alpha-D-glucopyranuronate

A method for the preparation of methyl 4-O-methyl-alpha-D-glucopyranuronate and its single crystal X-ray structure determination are reported. The molecule adopts an almost ideal (4)C1 ((degree)C3) conformation.     

Surface Characterization of Sepiolite by Inverse Gas Chromatography

Inverse gas chromatography (IGC) was applied to characterize the surface of sepiolite. The adsorption thermodynamic parameters (the standard enthalpy (ΔH ⁰), entropy (ΔS ⁰) and free energy of adsorption (ΔG ⁰)), the dispersive component of the surface energy (γ _S ^d ), and the acid/base character of sepiolite surface were estimated by using the retention time of different non-polar and polar probes at infinite dilution region. The specific free energy of adsorption (ΔG ^sp ), the specific enthalpy of adsorption (ΔH ^sp ), and the specific entropy of adsorption (ΔS ^sp ) of polar probes on sepiolite were determined. ΔH ^sp were correlated with the donor and modified acceptor numbers of the probes to quantify the acidic K _A and the basic K _D parameters of the sepiolite surface. The values obtained for the parameters K _A and K _D indicated an acidic character for sepiolite surface.     

The aqueous cationic system sodium undecenoate-dodecyltrimethylammonium bromide at low concentration

The aqueous sodium undecenoate (SUD) –dodecyltrimethylammonium bromide (DTAB) catanionic system was studied at low concentration. The system did not precipitate, even at a 1:1 SUD:DTAB proportion, but showed the formation of a coacervate in a range of surfactant mixture compositions. Micelles have a preferential composition of 0.37 mole fraction of SUD. This behavior is attributed to the presence of the double bond at the distal extreme of the SUD molecule, which can form hydrogen bonds with water. Consequently, the –CH=CH₂ group is situated at the interface between the hydrocarbon micelle core and water, reducing the interfacial free energy. Structural computations demonstrate that the mentioned SUD proportion produces complete coverage of the micelle surface by the double bonds.     

Substituent-dependent reactivity in the photodimerization of N-substituted dibenz[b,f]azepines

The photoprocesses of a series of N-substituted dibenz[b,f]azepines (iminostilbenes) were studied by absorption and emission spectroscopy, by laser flash photolysis, and by preparative irradiation with NMR analysis. In solutions, 2pi+2pi photodimers of N-cyano and N-acyl dibenzazepines are formed via the triplet state upon acetone- or benzophenone-sensitized energy transfer. T-T absorption spectra were measured and absorption coefficients were determined. The triplet energy transfer is equally efficient for N-alkyl dibenzazepines, which do not dimerize. Excited states of npi* character in the latter cases are discussed to rationalize the different reactivities. In spite of negligible intersystem crossing of 21 dibenzazepine derivatives, photodimers of N-acyl and N-cyano dibenzazepines are formed upon direct excitation in concentrated solutions (0.01-0.1 mol dm(-3)) as well as in the solid state. A selective anti-configuration of the photodimers was found throughout.     

Analysis of Colubroidea snake venoms by liquid chromatography with mass spectrometry: evolutionary and toxinological implications

The evolution of the venomous function of snakes and the diversification of the toxins has been of tremendous research interest and considerable debate. It has become recently evident that the evolution of the toxins in the advanced snakes (Colubroidea) predated the evolution of the advanced, front-fanged delivery mechanisms. Historically, the venoms of snakes lacking front-fanged venom-delivery systems (conventionally grouped into the paraphyletic family Colubridae) have been largely neglected. In this study we used liquid chromatography with mass spectrometry (LC/MS) to analyze a large number of venoms from a wide array of species representing the major advanced snake clades Atractaspididae, Colubrinae, Elapidae, Homalopsinae, Natricinae, Psammophiinae, Pseudoxyrhophiinae, Xenodontinae, and Viperidae. We also present the first sequences of toxins from Azemiops feae as well as additional toxin sequences from the Colubrinae. The large body of data on molecular masses and retention times thus assembled demonstrates a hitherto unsuspected diversity of toxins in all lineages, having implications ranging from clinical management of envenomings to venom evolution to the use of isolated toxins as leads for drug design and development. Although definitive assignment of a toxin to a protein family can only be done through demonstrated structural studies such as N-terminal sequencing, the molecular mass data complemented by LC retention information, presented here, do permit formulation of reasonable hypotheses concerning snake venom evolution and potential clinical effects to a degree not possible till now, and some hypotheses of this kind are proposed here. The data will also be useful in biodiscovery.